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1.  Crystallographic Identification of Lipid as an Integral Component of the Epitope of HIV Broadly Neutralizing Antibody 4E10 
Immunity  2016;44(1):21-31.
SUMMARY
Numerous studies of the anti-HIV-1 envelope glycoprotein 41 (gp41) broadly neutralizing antibody 4E10 suggest that 4E10 also interacts with membrane lipids, but the antibody regions contacting lipids and its orientation with respect to the viral membrane are unknown. Vaccine immunogens capable of re-eliciting these membrane proximal external region (MPER)-like antibodies may require a lipid component to be successful. We performed a systematic crystallographic study of lipid binding to 4E10 to identify lipids bound by the antibody and the lipid-interacting regions. We identified phosphatidic acid, phosphatidylglycerol, and glycerol phosphate as specific ligands for 4E10 in the crystal structures. 4E10 used its CDRH1 loop to bind the lipid head groups, while its CDRH3 interacted with the hydrophobic lipid tails. Identification of the lipid binding sites on 4E10 may aid design of immunogens for vaccines that include a lipid component in addition to the MPER on gp41 for generation of broadly neutralizing antibodies.
Graphical Abstract
doi:10.1016/j.immuni.2015.12.001
PMCID: PMC4720917  PMID: 26777395
2.  Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies 
Science immunology  2016;1(1):aaf7962.
A subset of bovine antibodies have an exceptionally long third heavy-chain complementarity determining region (CDR H3) that is highly variable in sequence and includes multiple cysteines. These long CDR H3s (up to 69 residues) fold into a long stalk atop which sits a knob domain that is located far from the antibody surface. Three new bovine Fab crystal structures have been determined to decipher the conserved and variable features of ultralong CDR H3s that lead to diversity in antigen recognition. Despite high sequence variability, the stalks adopt a conserved β-ribbon structure, while the knob regions share a conserved β-sheet that serves as a scaffold for two connecting loops of variable length and conformation, as well as one conserved disulfide. Variation in patterns and connectivity of the remaining disulfides contribute to the knob structural diversity. The unusual architecture of these ultralong bovine CDR H3s for generating diversity is unique in adaptive immune systems.
doi:10.1126/sciimmunol.aaf7962
PMCID: PMC5000368  PMID: 27574710
3.  Affinity maturation of a potent family of HIV antibodies is primarily focused on accommodating or avoiding glycans 
Immunity  2015;43(6):1053-1063.
SUMMARY
The high mannose patch on the HIV-1 Envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet differences evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provide insights to advance design of HIV vaccine candidates.
doi:10.1016/j.immuni.2015.11.007
PMCID: PMC4692269  PMID: 26682982
4.  Dynamic Local Polymorphisms in the Gbx1 Homeodomain Induced by DNA Binding 
Structure (London, England : 1993)  2016;24(8):1372-1379.
SUMMARY
The Gastrulation Brain Homeobox 1 (Gbx1) gene encodes the Gbx1 homeodomain that targets TAATTA motifs in dsDNA. Residues Glu17 and Arg52 in Gbx1 form a salt bridge, which is preserved in crystal structures and MD simulations of homologous homeodomain–DNA complexes. In contrast, our NMR studies show that DNA-binding to Gbx1 induces dynamic local polymorphisms, which include breaking of the Glu17–Arg52 salt bridge. To study this interaction, we produced a variant with Glu17Arg and Arg52Glu mutations, which exhibited the same fold as the wild-type protein, but a two-fold reduction in affinity for dsDNA. Analysis of the NMR structures of the Gbx1 homeodomain in the free form, the Gbx1[E17R,R52E] variant, and a Gbx1 homeodomain–DNA complex showed that stabilizing interactions of the Arg52 side chain with the DNA backbone are facilitated by transient breakage of the Glu17–Arg52 salt bridge in the DNA-bound Gbx1.
doi:10.1016/j.str.2016.05.013
PMCID: PMC5109927  PMID: 27396829
Binding affinity; NMR structure determination; J-UNIO; protein; DNA complex
5.  HMGB1 activates proinflammatory signaling via TLR5 leading to allodynia 
Cell reports  2016;17(4):1128-1140.
SUMMARY
Infectious and sterile inflammatory diseases are correlated with increased levels of high mobility group box-1 (HMGB1) in tissues and serum. Extracellular HMGB1 is known to activate toll-like receptors (TLRs) 2, 4 and RAGE (receptor for advanced glycation endproducts) in inflammatory conditions. Here we find that TLR5 is also an HMGB1 receptor that was previously overlooked due to lack of functional expression in the cell lines usually used for studying TLR signaling. HMGB1 binding to TLR5 initiates NF-κB signaling pathway activation in a MyD88-dependent manner, resulting in proinflammatory cytokine production and pain enhancement in vivo. Biophysical and in vitro results highlight an essential role for the C-terminal tail region of HMGB1 in facilitating interactions with TLR5. These results suggest that HMGB1-modulated TLR5 signaling is responsible for pain hypersensitivity.
Graphical abstract
doi:10.1016/j.celrep.2016.09.076
PMCID: PMC5087801  PMID: 27760316
6.  Adaptive Mutations Alter Antibody Structure and Dynamics During Affinity Maturation 
Biochemistry  2015;54(11):2085-2093.
While adaptive mutations can confer proteins with new function via the introduction or optimization of reactive centers, or other structural changes, a role for the optimization of protein dynamics also seems likely but has been more difficult to evaluate. Antibody (Ab) affinity maturation is an example of adaptive evolution wherein the adaptive mutations may be identified and Abs may be raised to specific targets that facilitate the characterization of protein dynamics. Here, we report the characterization of three affinity matured Abs that evolved from a common germline precursor to bind the chromophoric antigen (Ag), 8-methoxypyrene-1,3,6-trisulfonate (MPTS). In addition to characterizing the sequence, molecular recognition, and structure of each Ab, we characterized the dynamics of each complex by determining their mechanical response to an applied force via 3-pulse photon echo peak shift (3PEPS) spectroscopy and deconvoluting the response into elastic, anelastic, and plastic components. We find that for one Ab, affinity maturation was accomplished via the introduction of a single functional group that mediates a direct contact with MPTS and which results in a complex with little anelasticity or plasticity. In the other two cases, more mutations were introduced, but none directly contact MPTS, and while their effects on structure are subtle, their effects on anelasticity and plasticity are significant, with the level of plasticity correlated with specificity, suggesting that the optimization of protein dynamics may have contributed to affinity maturation. A similar optimization of structure and dynamics may contribute to the evolution of other proteins.
Graphical Abstract
doi:10.1021/bi501417q
PMCID: PMC5061043  PMID: 25756188
7.  Complete epitopes for vaccine design derived from a crystal structure of the broadly neutralizing antibodies PGT128 and 8ANC195 in complex with an HIV-1 Env trimer 
The crystal structure of the broadly neutralizing antibodies 8ANC195 and PGT128 bound to an HIV-1 envelope trimer has been determined. Structural and binding analyses have elucidated the full epitopes for these antibodies in the context of the intact viral glycoprotein, providing improved templates for HIV-1 vaccine design.
The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody–gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design.
doi:10.1107/S1399004715013917
PMCID: PMC4601371  PMID: 26457433
HIV-1; SOSIP gp140; broadly neutralizing antibodies; vaccine design; glycans
8.  Meta‐analysis of clinical metabolic profiling studies in cancer: challenges and opportunities 
EMBO Molecular Medicine  2016;8(10):1134-1142.
Abstract
Cancer cell metabolism has received increasing attention. Despite a boost in the application of clinical metabolic profiling (CMP) in cancer patients, a meta‐analysis has not been performed. The primary goal of this study was to assess whether public accessibility of metabolomics data and identification and reporting of metabolites were sufficient to assess which metabolites were consistently altered in cancer patients. We therefore retrospectively curated data from CMP studies in cancer patients published during 5 recent years and used an established vote‐counting method to perform a semiquantitative meta‐analysis of metabolites in tumor tissue and blood. This analysis confirmed well‐known increases in glycolytic metabolites, but also unveiled unprecedented changes in other metabolites such as ketone bodies and amino acids (histidine, tryptophan). However, this study also highlighted that insufficient public accessibility of metabolomics data, and inadequate metabolite identification and reporting hamper the discovery potential of meta‐analyses of CMP studies, calling for improved standardization of metabolomics studies.
doi:10.15252/emmm.201606798
PMCID: PMC5048364  PMID: 27601137
cancer; meta‐analysis; metabolic profiling; metabolomics; Cancer; Metabolism; Systems Medicine
9.  HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies 
Immunity  2016;45(3):483-496.
Summary
Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.
Highlights
•Developed mammalian cell display to design germline-targeting native-like trimers•Germline-targeting trimers retain native-like antigenicity and structure•Germline-targeting trimers prime glycan-dependent HIV bnAb responses in knockin mice•Designed boosting schemes intended to induce bnAbs
Elicitation of broadly neutralizing antibodies (bnAbs) is a critical HIV vaccine goal. Steichen et al. have developed immunogens that prime germline-precursor B cells for the bnAb PGT121 and can therefore initiate bnAb induction. The authors have also designed boosting immunogens to shepherd the antibody maturation to develop bnAbs.
doi:10.1016/j.immuni.2016.08.016
PMCID: PMC5040827  PMID: 27617678
10.  Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A, B and C 
PLoS Pathogens  2016;12(9):e1005864.
We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the env genes of four isolates of human immunodeficiency virus type 1 (HIV-1); specifically BG505 (clade A), B41 (clade B), CZA97 (clade C) and DU422 (clade C). The various trimers were delivered either simultaneously (as a mixture of clade A + B trimers) or sequentially over a 73-week period. Autologous, Tier-2 neutralizing antibody (NAb) responses were generated to the clade A and clade B trimers in the bivalent mixture. When delivered as boosting immunogens to rabbits immunized with the clade A and/or clade B trimers, the clade C trimers also generated autologous Tier-2 NAb responses, the CZA97 trimers doing so more strongly and consistently than the DU422 trimers. The clade C trimers also cross-boosted the pre-existing NAb responses to clade A and B trimers. We observed heterologous Tier-2 NAb responses albeit inconsistently, and with limited overall breath. However, cross-neutralization of the clade A BG505.T332N virus was consistently observed in rabbits immunized only with clade B trimers and then boosted with clade C trimers. The autologous NAbs induced by the BG505, B41 and CZA97 trimers predominantly recognized specific holes in the glycan shields of the cognate virus. The shared location of some of these holes may account for the observed cross-boosting effects and the heterologous neutralization of the BG505.T332N virus. These findings will guide the design of further experiments to determine whether and how multiple Env trimers can together induce more broadly neutralizing antibody responses.
Author Summary
Native-like SOSIP trimers are a platform for development of immunogens aimed at inducing broadly neutralizing antibodies and, hence, a possible vaccine against HIV-1 infection. No previous study has reported on immune responses to more than one such trimer. Here, we assess how rabbits respond to immunization with two or three different trimers, based on virus sequences from HIV-1 clades A, B and C, to gain insights into whether each is immunogenic under various regimens. We find that autologous Tier-2 neutralizing antibody responses can be raised against each trimer immunogen, whether they are delivered simultaneously or sequentially. We also observed some boosting of the neutralization response to the first trimer when a second trimer was administered later. Cross-reactive neutralizing antibodies were seen but only sporadically. We also found that the key immunogenic epitopes on the Env trimers involved holes in the glycan shield, which normally protects the virus from antibody binding. These various findings will guide the design of future experiments in animals and eventually in humans.
doi:10.1371/journal.ppat.1005864
PMCID: PMC5023125  PMID: 27627672
12.  Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies 
Cell  2016;166(6):1459-1470.e11.
Summary
Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.
Graphical Abstract
Highlights
•Designed boosting immunogens to follow a VRC01-class germline-targeting prime•Boosted Abs selectively incorporate VRC01-class somatic mutations•Sequential boosts elicit VRC01-class mAbs with broad neutralization of N276A viruses•Vaccine-elicited VRC01-class mAbs neutralize one fully native HIV isolate
Development of boosting HIV envelope immunogens designed to guide the genetic and functional maturation of VRC01-class bnAbs provides a roadmap for the design of an effective HIV vaccine.
doi:10.1016/j.cell.2016.08.005
PMCID: PMC5018249  PMID: 27610570
13.  Holes in the glycan shield of the native HIV envelope are a target of trimer-elicited neutralizing antibodies 
Cell reports  2016;16(9):2327-2338.
SUMMARY
A major advance in the search for an HIV vaccine has been the development of a near-native Envelope trimer (BG505 SOSIP.664) that can induce robust autologous Tier 2 neutralization. Here, potently neutralizing monoclonal antibodies (nAbs) from rabbits immunized with BG505 SOSIP.664 are shown to recognize an immunodominant region of gp120 centered on residue 241. Residue 241 occupies a hole in the glycan defenses of the BG505 isolate with fewer than 3% of global isolates lacking a glycan site at this position. However, at least one conserved glycan site is missing in 89% of viruses, suggesting the presence of glycan holes in a majority of HIV isolates. Indeed, serum evidence is consistent with targeting of holes in natural infection. The immunogenic nature of breaches in the glycan shield has been under-appreciated in previous attempts to understand autologous neutralizing antibody responses and has important potential consequences for HIV vaccine design.
eTOC Blurb
This study describes monoclonal rabbit antibodies elicited by the HIV immunogen BG505 SOSIP.664. Previous reports showed high titer serum neutralization against BG505 virus but the epitope remained elusive. McCoy et al. show the neutralizing epitope is a breach in HIV’s glycan shield and suggest other HIV strains contain similar holes.
doi:10.1016/j.celrep.2016.07.074
PMCID: PMC5007210  PMID: 27545891
14.  Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch 
Immunity  2016;44(5):1215-1226.
SUMMARY
The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ~11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.
Graphical Abstract
doi:10.1016/j.immuni.2016.04.016
PMCID: PMC5003182  PMID: 27192579
15.  Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design 
PLoS Pathogens  2016;12(8):e1005815.
An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens.
Author Summary
Many HIV vaccine design efforts aim to elicit so-called broadly neutralizing antibodies that bind and neutralize diverse strains and subtypes of the virus. However, these efforts are guided by very unusual antibodies isolated from HIV-infected individuals. These antibodies have rare features that limit their use as direct vaccine templates, because it is unlikely that any vaccine could consistently elicit similar antibodies. We engineered HIV broadly neutralizing antibodies that minimized these rare features and may therefore serve as better leads for HIV vaccine design. Antibodies generally gain affinity for their target epitope by accumulating mutations in a natural process of maturation. Figuring out how to use vaccines to elicit particular kinds of antibodies, with particular kinds of helpful mutations, is a major unsolved challenge for vaccine design. We were able to determine which mutations in our new antibodies are most important and which epitope structures are needed to induce those mutations. This analysis allowed us to deduce a logical strategy, which remains to be tested, for how to guide the maturation of these types of antibodies by vaccination. We propose that this reductionist approach to vaccine design, guided by molecular structure and engineering-oriented to allow for optimization, has promise for designing vaccines against HIV and many other pathogens.
doi:10.1371/journal.ppat.1005815
PMCID: PMC4999182  PMID: 27560183
16.  Structure of hepatitis C virus envelope glycoprotein E1 antigenic site 314-324 in complex with antibody IGH526 
Journal of molecular biology  2015;427(16):2617-2628.
Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral “spike” of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies, are available for study of this essential HCV glycoprotein. A human MAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates and, therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that MAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314-324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen.
Graphical abstract
doi:10.1016/j.jmb.2015.06.012
PMCID: PMC4523428  PMID: 26135247
HCV; E1 envelope glycoprotein; IGH526; vaccine design
17.  H7N9 influenza virus neutralizing antibodies that possess few somatic mutations 
The Journal of Clinical Investigation  null;126(4):1482-1494.
Avian H7N9 influenza viruses are group 2 influenza A viruses that have been identified as the etiologic agent for a current major outbreak that began in China in 2013 and may pose a pandemic threat. Here, we examined the human H7-reactive antibody response in 75 recipients of a monovalent inactivated A/Shanghai/02/2013 H7N9 vaccine. After 2 doses of vaccine, the majority of donors had memory B cells that secreted IgGs specific for H7 HA, with dominant responses against single HA subtypes, although frequencies of H7-reactive B cells ranged widely between donors. We isolated 12 naturally occurring mAbs with low half-maximal effective concentrations for binding, 5 of which possessed neutralizing and HA-inhibiting activities. The 5 neutralizing mAbs exhibited narrow breadth of reactivity with influenza H7 strains. Epitope-mapping studies using neutralization escape mutant analysis, deuterium exchange mass spectrometry, and x-ray crystallography revealed that these neutralizing mAbs bind near the receptor-binding pocket on HA. All 5 neutralizing mAbs possessed low numbers of somatic mutations, suggesting the clones arose from naive B cells. The most potent mAb, H7.167, was tested as a prophylactic treatment in a mouse intranasal virus challenge study, and systemic administration of the mAb markedly reduced viral lung titers.
doi:10.1172/JCI85317
PMCID: PMC4811156  PMID: 26950424
18.  Fatty acid oxidation is required for the respiration and proliferation of malignant glioma cells 
Neuro-Oncology  2016;19(1):43-54.
Background.
Glioma is the most common form of primary malignant brain tumor in adults, with approximately 4 cases per 100 000 people each year. Gliomas, like many tumors, are thought to primarily metabolize glucose for energy production; however, the reliance upon glycolysis has recently been called into question. In this study, we aimed to identify the metabolic fuel requirements of human glioma cells.
Methods.
We used database searches and tissue culture resources to evaluate genotype and protein expression, tracked oxygen consumption rates to study metabolic responses to various substrates, performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates, and employed an animal model of malignant glioma to evaluate a new therapeutic intervention.
Results.
We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition, we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover, inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma.
Conclusions.
Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition, the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice.
doi:10.1093/neuonc/now128
PMCID: PMC5193020  PMID: 27365097
etomoxir; fatty acid oxidation; glioblastoma; glioma; metabolism
19.  Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability 
Nature Communications  2016;7:12040.
The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.
A major goal of HIV-1 vaccine development is to produce antigens that can induce broadly neutralizing antibodies. Here the authors examine the underlying causes of HIV-1 envelope metastability and design uncleaved, prefusion-optimized gp140 trimers with potential for use as HIV-1 vaccine antigens.
doi:10.1038/ncomms12040
PMCID: PMC4931249  PMID: 27349805
20.  Presenting native-like trimeric HIV-1 antigens with self-assembling nanoparticles 
Nature Communications  2016;7:12041.
Structures of BG505 SOSIP.664 trimer in complex with broadly neutralizing antibodies (bNAbs) have revealed the critical role of trimeric context for immune recognition of HIV-1. Presentation of trimeric HIV-1 antigens on nanoparticles may thus provide promising vaccine candidates. Here we report the rational design, structural analysis and antigenic evaluation of HIV-1 trimer-presenting nanoparticles. We first demonstrate that both V1V2 and gp120 can be presented in native-like trimeric conformations on nanoparticles. We then design nanoparticles presenting various forms of stabilized gp140 trimer based on ferritin and a large, 60-meric E2p that displays 20 spikes mimicking virus-like particles (VLPs). Particle assembly is confirmed by electron microscopy (EM), while antigenic profiles are generated using representative bNAbs and non-NAbs. Lastly, we demonstrate high-yield gp140 nanoparticle production and robust stimulation of B cells carrying cognate VRC01 receptors by gp120 and gp140 nanoparticles. Together, our study provides an arsenal of multivalent immunogens for HIV-1 vaccine development.
The development of native-like envelope trimers has been a major focus in the efforts to produce HIV vaccines. Here the authors demonstrate the production and characterization of virus-like nanoparticles displaying trimeric HIV-1 antigens with the potential to elicit broadly neutralizing antibodies.
doi:10.1038/ncomms12041
PMCID: PMC4931238  PMID: 27349934
21.  A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis elegans 
Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small molecules, represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe 4, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe 4 comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the timescale required for cellular imaging, but does red shift the emission maximum of probe 4 in comparison to its non-fluorosulfated analog. We demonstrate that probe 4 is dark in aqueous buffers, whereas the TTR•4 complex exhibits a fluorescence emission maximum at 481 nm. The addition of probe 4 to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live Caenorhabditis elegans expressing human TTR transgenically and treated with probe 4 display TTR•4 fluorescence in macrophage-like coelomocytes. An analog of fluorosulfate probe 4 does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analog suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes.
doi:10.1021/jacs.5b03042
PMCID: PMC4472559  PMID: 26051248
22.  Structural constraints determine the glycosylation of HIV-1 envelope trimers 
Cell reports  2015;11(10):1604-1613.
A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system but, paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity-purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles’ heel that can be exploited for bNAb recognition and vaccine design.
doi:10.1016/j.celrep.2015.05.017
PMCID: PMC4555872  PMID: 26051934
23.  Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry 
Journal of Chromatography. a  2016;1449:141-155.
Highlights
•An SFC method has been developed for the analysis of polar urinary metabolites.•12 stationary phases, 9 modifier additives and 3 temperatures were evaluated.•DIOL and 2-PIC columns provide highest peak capacity and overall resolution.•Ammonium formate, ammonium hydroxide and water considerably improved separation.•Alkylamine additives should be strongly considered for polar SFC-UV analysis.
Supercritical fluid chromatography (SFC) is frequently used for the analysis and separation of non-polar metabolites, but remains relatively underutilised for the study of polar molecules, even those which pose difficulties with established reversed-phase (RP) or hydrophilic interaction liquid chromatographic (HILIC) methodologies. Here, we present a fast SFC-MS method for the analysis of medium and high-polarity (−7 ≤ cLogP ≤ 2) compounds, designed for implementation in a high-throughput metabonomics setting. Sixty polar analytes were first screened to identify those most suitable for inclusion in chromatographic test mixtures; then, a multi-dimensional method development study was conducted to determine the optimal choice of stationary phase, modifier additive and temperature for the separation of such analytes using SFC. The test mixtures were separated on a total of twelve different column chemistries at three different temperatures, using CO2-methanol-based mobile phases containing a variety of polar additives. Chromatographic performance was evaluated with a particular emphasis on peak capacity, overall resolution, peak distribution and repeatability. The results suggest that a new generation of stationary phases, specifically designed for improved robustness in mixed CO2-methanol mobile phases, can improve peak shape, peak capacity and resolution for all classes of polar analytes. A significant enhancement in chromatographic performance was observed for these urinary metabolites on the majority of the stationary phases when polar additives such as ammonium salts (formate, acetate and hydroxide) were included in the organic modifier, and the use of water or alkylamine additives was found to be beneficial for specific subsets of polar analytes. The utility of these findings was confirmed by the separation of a mixture of polar metabolites in human urine using an optimised 7 min gradient SFC method, where the use of the recommended column and co-solvent combination resulted in a significant improvement in chromatographic performance.
doi:10.1016/j.chroma.2016.04.040
PMCID: PMC4927693  PMID: 27143232
SFC; Method development; Polar; Stationary phase; Modifier; Additive
24.  Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers 
Journal of Virology  2015;89(23):12189-12210.
ABSTRACT
We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components.
IMPORTANCE Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.
doi:10.1128/JVI.01768-15
PMCID: PMC4645310  PMID: 26311893
25.  HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen 
Science (New York, N.Y.)  2016;351(6280):1458-1463.
Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.
doi:10.1126/science.aad9195
PMCID: PMC4872700  PMID: 27013733

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